JP4929700B2 - Production method of carboxylic acid - Google Patents

Description

  The present invention relates to a method for producing carboxylic acid and the like.

  Conventionally, a method of hydrolyzing cyanohydrin or the like is used to produce hydroxycarboxylic acid or the like by a chemical synthesis method. Also, industrially, a method using sulfuric acid as a catalyst is known. Also known are methods for hydrolyzing hydroxynitrile compounds by the action of microorganisms to convert them into the corresponding hydroxycarboxylic acids (see, for example, Patent Document 1, Patent Document 2, and Patent Document 3).

  However, in the method using sulfuric acid as a catalyst, as a result of the reaction between the hydroxynitrile compound and sulfuric acid, the target product, hydroxycarboxylic acid, and an equimolar amount of ammonium sulfate are by-produced. In addition, in the method of producing a corresponding hydroxycarboxylic acid compound from a hydroxynitrile compound using a microorganism, the enzyme activity retained by the microorganism is inhibited by a decomposition product from the hydroxynitrile compound or by-product. Therefore, it is necessary to process a large amount of ammonium salt, which increases the manufacturing cost and is complicated.

Japanese Patent Publication No.58-15120 Japanese Patent Laid-Open No. 2-84198 Japanese Patent Laid-Open No. 4-40898

Provided is a production method using a microorganism of carboxylic acid which does not inhibit enzyme activity or cause by-production and treatment of a large amount of ammonium salt.

That is, the present invention
1. Formula (1):

(Wherein R ¹ represents hydrogen, an alkyl group, or a hydroxyl group, and R ² represents an alkyl group or an aryl group.)
Or an alcohol compound represented by formula (2):

(Wherein R ¹ and R ² represent the same meaning as described above.)
Or a microbial cell belonging to a microorganism belonging to Rhodococcus rhodochrous or Rhodococcus sp. Having the ability to convert the alcohol compound or the aldehyde compound into a corresponding carboxylic acid compound. Formula (3), characterized in that a treated bacterial cell is allowed to act:

(Wherein R ¹ and R ² represent the same meaning as described above.)
A method for producing a carboxylic acid compound represented by the formula (hereinafter also referred to as the present invention production method);
2. 2. The production method according to item 1 above, wherein the microorganism is Rhodococcus rhodochrous ATCC15610 strain or Rhodococcus sp. ATCC19148 strain;
3. The production method according to 1 or 2 above, wherein the microorganism is a microorganism produced in a medium supplemented with a primary or secondary alcohol;
4). The production method according to item 3, wherein the primary or secondary alcohol to be added is a primary or secondary alcohol having 1 to 5 carbon atoms;
5. 4. The production according to item 3 above, wherein the primary or secondary alcohol to be added is methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-methyl-1-propanol, or 2,2-dimethyl-1-propanol. Law;
Etc. are provided.

  According to the present invention, carboxylic acid can be advantageously produced industrially using microorganisms without the risk of enzyme activity being inhibited or the production and treatment of a large amount of ammonium salt.

Hereinafter, the production method of the present invention will be described.

In the alcohol compound represented by the formula (1), the aldehyde compound represented by the formula (2), and the carboxylic acid compound represented by the formula (3), examples of the alkyl group represented by R ¹ include, for example, methyl group, ethyl Examples thereof include alkyl groups having 1 to 8 carbon atoms such as a group, propyl group, butyl group, pentyl group, hexyl group, heptyl group and octyl group.
Examples of the alkyl group represented by R ² include 1-7 carbon atoms such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a t-butyl group, a pentyl group, a hexyl group, and a heptyl group. The alkyl group of is illustrated.
Examples of the aryl group represented by R ² include aryl groups having 6 to 20 carbon atoms such as a phenyl group, a tolyl group, and a naphthyl group.
Specific examples of the alcohol compound represented by the formula (1) include 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, 1-heptanol, 1-octanol, 1-nonanol, and 1,2-butanediol. 1,2-pentanediol, 1,2-hexanediol, 1,2-heptanediol, 1,2-octanediol, 1,2-nonanediol, 2-methyl-1-propanol, 2-methyl-1- Butanol, 2-methyl-1-pentanol, 2-methyl-1-hexanol, 2-methyl-1-heptanol, 2-methyl-1-octanol, 2-methyl-1-nonanol, 3-phenyl-1-propanol 4-phenyl-1-butanol, 5-phenyl-1-pentanol, 6-phenyl-1-hexanol, etc. . Specific examples of the aldehyde compound represented by the formula (2) include 1-propionaldehyde, 1-butanal, 1-pentanal, 1-hexanal, 1-heptanal, 1-octanal, 1-nonanal, 2- Examples include methylpropanal, 2-methylbutyraldehyde, 2-methylvaleraldehyde, 3-phenylpropionaldehyde and the like. The carboxylic acid compounds produced are specifically propionic acid, n-butyric acid, n-valeric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, isobutyric acid, 2-methyl-butyric acid, 2-methylpentanoic acid , 2-methylhexanoic acid, 2-methylheptanoic acid, 2-methyloctanoic acid, 2-methylnonanoic acid, 3-phenylpropionic acid, 4-phenylbutyric acid, 5-phenylpentanoic acid, 6-phenylhexanoic acid, etc. The

  In addition, the carboxylic acid compound represented by the formula (3) corresponding to the alcohol compound represented by the formula (1) or the aldehyde compound represented by the formula (2), which is produced by the production method of the present invention and recovered from the reaction solution May be in the form of a salt.

  Rhodococcus rhodochrous or Rhodococcus sp. Having the ability to convert the alcohol compound of formula (1) or the aldehyde compound of formula (2) used in the production method of the present invention into the corresponding carboxylic acid, etc. Specific examples of the microbial cells such as the above-mentioned microorganisms or the processed microbial cells include, for example, Rhodococcus rhodochrous ATCC15610 and Rhodococcus sp. ATCC19148.

When the alcohol compound of the formula (1) or the aldehyde compound of the formula (2) in the formula (1) is brought into contact with such a microbial cell or a treated product of the microbial cell, when R ₁ is a hydroxyl group, the primary hydroxyl group is preferentially oxidized. it can. Here, “preferentially oxidizable” means that oxidation of the primary hydroxyl group proceeds preferentially over oxidation of the secondary hydroxyl group of the dihydroxy compound.

  Microorganisms such as Rhodococcus rhodochrous or Rhodococcus sp. Having the ability to convert the alcohol compound of formula (1) or the aldehyde compound of formula (2) into the corresponding carboxylic acid are a carbon source, What is necessary is just to culture | cultivate using the culture medium for culturing various microorganisms containing a nitrogen source, organic salt, inorganic salt, etc. suitably.

Examples of the carbon source contained in the medium include glucose, sucrose, glycerol, starch, alcohol, organic acid and molasses. As the alcohol, a primary or secondary alcohol having 1 to 5 carbon atoms is particularly preferable. For example, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-methyl-1-propanol, 2, 2-dimethyl-1-propanol is mentioned. Reactivity can be enhanced by culturing the microorganism in a medium to which alcohol has been added.
Nitrogen sources include, for example, yeast extract, meat extract, peptone, casamino acid, malt extract, soy flour, corn steep liquor, cottonseed flour, dry yeast, ammonium sulfate and sodium nitrate, organic Examples of the salt and inorganic salt include sodium chloride, potassium chloride, sodium carbonate, 1 potassium phosphate, 2 potassium phosphate, calcium carbonate, ammonium acetate, magnesium sulfate, copper sulfate, zinc sulfate, ferrous sulfate and cobalt chloride. Is mentioned.

  Examples of the culture method include solid culture and liquid culture (test tube culture, flask culture, jar fermenter culture, etc.).

  The culture temperature and the pH of the culture solution are not particularly limited as long as the microorganism grows. For example, the culture temperature is in the range of about 15 to 45 ° C., and the pH of the culture solution is about 4 to 8. A range can be mentioned. The culture time can be appropriately selected depending on the culture conditions, but is usually about 1 to 7 days.

  The bacterial cells thus obtained can be used as they are in the production method of the present invention. As a method of using the cells of this microorganism as they are, (1) a method of using the culture solution as it is, (2) collecting the cells by centrifugation of the culture solution, and collecting the collected cells (if necessary, a buffer solution Or a method using a wet cell after washing with water.

  In the production method of the present invention, the processed product of the cells can also be used. Examples of the treated cells include cells obtained by culturing cells treated with an organic solvent (acetone, ethanol, etc.), freeze-dried or alkali-treated, or cells physically or Enzymatically crushed or crude enzyme separated and extracted from these can be mentioned. Furthermore, what was fixed by the well-known method is included in a microbial cell processed material after giving the said process.

  The production method of the present invention is usually performed in the presence of water. The water in this case may be in the form of a buffer solution. Examples of the buffer used in the buffer include alkali metal salts of phosphoric acid such as sodium phosphate and potassium phosphate, and alkali metal salts of acetic acid such as sodium acetate and potassium acetate.

  In addition, the production method of the present invention can be carried out using a hydrophobic organic solvent in the presence of water and a hydrophobic organic solvent. Examples of the hydrophobic organic solvent used in this case include esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, ethyl propionate and butyl propionate, n-butyl alcohol, n-amyl alcohol, n- Alcohols such as octyl alcohol, aromatic hydrocarbons such as benzene, toluene and xylene, ethers such as diethyl ether, diisopropyl ether and methyl-t-butyl ether, and halogenated hydrocarbons such as chloroform and 1,2-dichloroethane And mixtures thereof.

  Moreover, this invention manufacturing method can also be performed in presence of water and a hydrophilic organic solvent using a hydrophilic organic solvent. Examples of the hydrophilic organic solvent used in this case include alcohols such as methanol and ethanol, ketones such as acetone, ethers such as dimethoxyethane, tetrahydrofuran and dioxane, and mixtures thereof.

  The production method of the present invention is usually carried out within a pH range of 3 to 10 in the aqueous layer, but may be appropriately changed within the range in which the reaction proceeds.

  The production method of the present invention is usually performed within a range of about 0 to 60 ° C., but may be appropriately changed within a range in which the reaction proceeds.

  The production method of the present invention is usually performed within a range of about 0.5 hours to about 10 days. The end point of the reaction can be confirmed by measuring the amount of the sulfur-containing dihydroxy compound in the reaction solution, for example, by liquid chromatography, gas chromatography or the like after the addition of the sulfur-containing dihydroxy compound as the raw material compound. it can.

  The concentration of the alcohol compound or aldehyde compound, which is a raw material compound in the production method of the present invention, is usually 50% (w / v) or less, in order to keep the concentration of the alcohol compound or aldehyde compound in the reaction system substantially constant. In addition, the alcohol compound or aldehyde compound may be continuously or sequentially added to the reaction system.

  In the production method of the present invention, for example, a sugar such as glucose, sucrose, or fructose, or a surfactant such as Triton X-100 (registered trademark) or Tween 60 (registered trademark) is added to the reaction system as necessary. You can also.

  After the completion of the reaction, the reaction solution may be subjected to usual post-treatment such as organic solvent extraction, filtration, and concentration to recover the alcohol compound or the carboxylic acid compound corresponding to the aldehyde compound from the reaction solution. The recovered carboxylic acid compound can be further purified by column chromatography, distillation or the like, if necessary.

EXAMPLES Hereinafter, although the Example manufacturing method is demonstrated in detail by an Example, this invention manufacturing method is not limited to these examples.

Example 1 (Production Example (No. 1) of Carboxylic Acid Compound from Alcohol Compound or Aldehyde Compound According to the Production Method of the Present Invention)
Sterilized medium (glucose 20 g, polypeptone 5 g, yeast extract 3 g, meat extract 3 g, ammonium sulfate 0.2 g, potassium dihydrogen phosphate 1 g and magnesium sulfate heptahydrate 0.5 g was added to a test tube. After that, 5 ml of a solution adjusted to pH 7.0) was added, and Rhodococcus rhodochrous ATCC15610 strain or Rhodococcus sp. ATCC19148 strain was inoculated therein. This was cultured with shaking at 30 ° C. under aerobic conditions. After culturing, the cells were separated by centrifugation to obtain viable cells. 1 ml of 0.1 M potassium phosphate buffer (pH 7) was placed in a screw-mouth test tube, and the above viable cells were added thereto, followed by suspension. After adding an alcohol compound or an aldehyde compound to the suspension to a final concentration of 1% (w / v), the resulting mixture was shaken at 30 ° C. for 24 hours.

After completion of the reaction, 0.5 ml of the reaction solution was sampled. After removing the cells from the sampling solution, the amount of the produced carboxylic acid was analyzed by liquid chromatography, or the sampling solution was acidified, extracted with ethyl acetate, and then analyzed by gas chromatography. The obtained results are shown in Table 1.
(Content analysis condition 1)
Column: Cadenza CD-C18 (4.6 mmφ × 15 cm, 3 μm) (manufactured by Imtakt)
Mobile phase: A liquid 0.1% trifluoroacetic acid aqueous solution, B liquid Methanol time (min) A liquid (%): B liquid (%)
0 80:20
10 80:20
20 50:50
30 50:50
30.1 80:20
Flow rate: 0.5 ml / min, column temperature: 40 ° C., detection: 220 nm
(Content analysis condition 2)
Column: DB-1 (0.53 mm × 30 m, 1.5 μm) (manufactured by J & W Scientific)
Column temperature: 70 ° C. (0 min) → 4 ° C./min→170° C. (0 min) → 30 ° C./min→290° C. (5 min)
Carrier gas: helium (flow rate: 7 ml / min), detector: FID, injection volume: 1 μl

Example 2 (Production Example of Carboxylic Acid Compound from Alcohol Compound or Aldehyde Compound According to the Production Method of the Present Invention (Part 2))
In a test tube, sterilized medium (1 g of 20 g 1-propanol, 5 g polypeptone, 3 g yeast extract, 3 g meat extract, 0.2 g ammonium sulfate, 1 g potassium dihydrogen phosphate and 0.5 g magnesium sulfate heptahydrate After the addition, 5 ml of a solution adjusted to pH 7.0) was added, and Rhodococcus rhodochrous ATCC15610 strain or Rhodococcus sp. ATCC19148 strain was inoculated therein. This was cultured with shaking at 30 ° C. under aerobic conditions. After culturing, the cells were separated by centrifugation to obtain viable cells. 1 ml of 0.1 M potassium phosphate buffer (pH 7) was placed in a screw-mouth test tube, and the above viable cells were added thereto, followed by suspension. After adding an alcohol compound or an aldehyde compound to the suspension to a final concentration of 1% (w / v), the resulting mixture was shaken at 30 ° C. for 24 hours.

  After completion of the reaction, 0.5 ml of the reaction solution was sampled. After removing the cells from the sampling solution, the amount of the produced carboxylic acid was analyzed by liquid chromatography, or the sampling solution was acidified, extracted with ethyl acetate, and then analyzed by gas chromatography. The obtained results are shown in Table 2.

(Content analysis condition 1)
Column: Cadenza CD-C18 (4.6 mmφ × 15 cm, 3 μm) (manufactured by Imtakt)
Mobile phase: A liquid 0.1% trifluoroacetic acid aqueous solution, B liquid Methanol time (min) A liquid (%): B liquid (%)
0 80:20
10 80:20
20 50:50
30 50:50
30.1 80:20
Flow rate: 0.5 ml / min Column temperature: 40 ° C
Detection: 220nm

(Content analysis condition 2)
Column: DB-1 (0.53 mm × 30 m, 1.5 μm) (manufactured by J & W Scientific)
Column temperature: 70 ° C. (0 min) → 4 ° C./min→170° C. (0 min) → 30 ° C./min→290° C. (5 min)
Carrier gas: helium (flow rate: 7 ml / min)
Detector: FID
Injection volume: 1μl

Example 3 (Production example of 2-hydroxybutyric acid from 1,2-butanediol by the production method of the present invention)
Sterilized medium (1 L of water, various alcohols 20 g shown in Table 3, 5 g of polypeptone, 3 g of yeast extract, 3 g of meat extract, 0.2 g of ammonium sulfate, 1 g of potassium dihydrogen phosphate and 7 water of magnesium sulfate) After adding 0.5 g of Japanese product, the pH was adjusted to 7.0) 5 ml), and Rhodococcus rhodochrous ATCC15610 strain or Rhodococcus sp. ATCC19148 strain was inoculated therein. This was cultured with shaking at 30 ° C. under aerobic conditions. After culturing, the cells were separated by centrifugation to obtain viable cells. 1 ml of 0.1 M potassium phosphate buffer (pH 7) was placed in a screw-mouth test tube, and the above viable cells were added thereto, followed by suspension. To the suspension, 1,2-butanediol was added to a final concentration of 1% (w / v)), and the resulting mixture was shaken at 30 ° C. for 24 hours.

After completion of the reaction, 0.5 ml of the reaction solution was sampled. After removing the cells from the sampling solution, the amount of the produced carboxylic acid was analyzed by liquid chromatography. The obtained results are shown in Table 3.
(Content analysis conditions)
Column: Cadenza CD-C18 (4.6 mmφ × 15 cm, 3 μm) (manufactured by Imtakt)
Mobile phase: A liquid 0.1% trifluoroacetic acid aqueous solution, B liquid Methanol time (min) A liquid (%): B liquid (%)
0 80:20
10 80:20
20 50:50
30 50:50
30.1 80:20
Flow rate: 0.5 ml / min Column temperature: 40 ° C
Detection: 220nm

According to the present invention, a carboxylic acid compound can be produced efficiently.

Claims (5)

In the absence of cyanic acid, formula (1):

(In the formula, R ¹ represents hydrogen, an alkyl group having 1 to 8 carbon atoms , or a hydroxyl group, and R ² represents an alkyl group having 1 to 7 carbon atoms or an aryl group having 6 to 20 carbon atoms .)
Or an alcohol compound represented by formula (2)

(Wherein R ¹ and R ² represent the same meaning as described above.)
A formula in which a microbial cell or a treated product of a microorganism belonging to Rhodococcus rhodochrous having the ability to convert the alcohol compound or the aldehyde compound into a corresponding carboxylic acid compound is allowed to act on the aldehyde compound represented by ( 3):

(Wherein R ¹ and R ² represent the same meaning as described above.)
And the microorganism acts on the alcohol compound represented by the formula (1) or the aldehyde compound represented by the formula (2) by acting on the microorganism or the treated product of the microorganism. A production method characterized by being a microorganism produced by culturing in a medium to which a primary or secondary alcohol is added before the treatment. In the absence of cyanic acid, formula (1):

(In the formula, R ¹ represents hydrogen, an alkyl group having 1 to 8 carbon atoms, or a hydroxyl group, and R ² represents an alkyl group having 1 to 7 carbon atoms or an aryl group having 6 to 20 carbon atoms.)
Or an alcohol compound represented by formula (2)

(Wherein R ¹ and R ² represent the same meaning as described above.)
Rhodococcus rhodochrous ATCC15610 strain or Rhodococcus sp. ATCC19148 strain having the ability to convert the alcohol compound or aldehyde compound into the corresponding carboxylic acid compound Formula (3) for acting a microbial cell or a processed microbial cell:

(Wherein R ¹ and R ² represent the same meaning as described above.)
And the microorganism acts on the alcohol compound represented by the formula (1) or the aldehyde compound represented by the formula (2) by acting on the microorganism or the treated product of the microorganism. A production method characterized by being a microorganism produced by culturing in a medium to which a primary or secondary alcohol is added before the treatment. R ¹ Is hydrogen, a methyl group or a hydroxyl group, and R ² The method according to claim 1 or 2, wherein is an alkyl group having 1 to 7 carbon atoms or a benzyl group. Primary or secondary alcohol, The method according to any one of claims 1 to 3 is a primary or secondary alcohol having 1 to 5 carbon atoms. Primary or secondary alcohol, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, claims 1 to 3 is 2-methyl-1-propanol or 2,2-dimethyl-1-propanol, The manufacturing method in any one of.